Respuestas fúngicas a señales ambientales: Funciones de las proteínas caro, opsa, cuta y acya en fusarium fujikuroi
- Autores: Jorge García Martínez
- Directores de la Tesis: Francisco Javier Ávalos Cordero (dir. tes.)
- Lectura: En la Universidad de Sevilla ( España ) en 2014
- Idioma: español
- Tribunal Calificador de la Tesis: Jesús Pla Alonso (presid.), Jesús de la Cruz Díaz (secret.), Eduardo R. Bejarano (voc.), M.ª Carmen Ruiz Roldán (voc.), Alfred Batschauer (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: Idus
- Resumen
The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. In the chapter 1 of this Thesis, we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments (probably fusarubin), reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, ¿acyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress, but no change in resistance to heavy metals or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group.
Survival of microorganisms in natural habitats depends on their ability to adapt to variations in osmotic conditions. In Chapter 2, we provide novel data on the molecular mechanism used by F. fujikuroi to overcome hyperosmotic stress. The research group formerly described the gene cut-1 of Neurospora crassa, encoding a protein of the haloacid dehalogenase family with an unknown function in the osmotic stress response. Here we report on the functional analysis of cutA, the cut-1 ortholog of F. fujikuroi. cutA mRNA levels increased transiently after exposure to 0.68 M NaCl and were reduced upon return to normal osmotic conditions; deletion of the gene resulted in a partial reduction in tolerance to osmotic stress. ¿cutA mutants contained much lower intracellular levels of glycerol than the wild-type, and did not exhibit the glycerol increase that follows an hyper-osmotic shock, as expected from the high osmolarity glycerol (HOG) response. cutA is linked and divergently transcribed with the putative glycerol dehydrogenase gene gldB, which showed the same regulation by osmotic shock. The intergenic cutA/gldB regulatory region contains putative stress-response elements conserved in other fungi, and both genes shared other regulatory features, such as induction by heat shock and by illumination. Photoinduction was also observed in the HOG response gene hogA, and was lost in mutants of the white collar gene wcoA. Previous data on glycerol production in Aspergillus spp. and features of the predicted CutA protein lead us to propose that F. fujikuroi produces glycerol from dihydroxyacetone, and that CutA is the enzyme involved in the synthesis of this precursor by dephosphorylation of dihydroxyacetone-3P.
The last Chapter of this Thesis is focused on the study of Fusarium rhodopsins, membrane-embedded photoreceptors using retinal as chromo-phore, found in all major taxonomic kingdoms. Rhodopsins play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton pumping activity and the possible effects of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Comparative germination analyses of conidia from a carO- mutant and its carO+ control strain showed a faster development of light-exposed carO- germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down conidia germination and early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological function for a fungal rhodopsin.
